Apurinic/apyrimidinic endonuclease (Ape1) is a multifunctional protein that is active in the removal of damaged or modified bases in the DNA base excision repair (BER) pathway and in the redox regulation of a variety of transcription factors including AP-1, Nf-kB, CREB/ATF and P53. Certain DNA-damaging agents induce the expression of Ape1. In addition, its expression appears to be cell cycle regulated. Increase in Ape1 expression is observed in certain types of cancers and also during wound healing. Interestingly, homozygous deletion of the mouse Ape1 results into both cellular and embryonic lethality. The reason for this lethality remains an unanswered question, as does the transcriptional regulation of this important repair protein. The long-term goal of this project is to examine the regulation of APE1 using the Chinese hamster gene as a model and to assess the effects of altered expression of APE1 in repair proficient and deficient cell lines. In the current application we propose to examine the factors involved in transcriptional control of chAPE1, the hamster homolog to the Ape1 and to identify the role(s) of the transcription factors involved. We will also examine chAPE1 up-regulation in response to DNA damaging agents and determine which site(s) are involved in this response. This is a natural extension of our current research. The first aim of the application is to examine the DNA-protein complexes formed by the various putative regulatory elements present in the 400 bp promoter region and their effect on chAPE1 gene regulation. We will investigate the effects of site-specific mutations on the formation of DNA-protein complexes. The second aim of the project is to identify specific DNA sequences to which factors from COH cells nuclear extracts bind. We will employ DNase I foot printing to identify the sequences. The third aim of the proposed research is to assess the effects of DNA damaging agents on the expression of the chAPE gene product. We will use either the SEAP or CAT reporter system and a transient expression system to examine chAPE1 expression after exposure to DNA damaging agents. The last aim of the proposed research is to generate stable transfected CHO-9 cell lines. These cell lines will be used to examine the role of different transcriptional regulatory motifs within an intact system. Both normal and mutated enhancer/ promoter constructs will be used.